Am J Hum Genet 36:1121-1134, 1984 Beta-Thalassemia in the Po Delta: Selection, Geography, and Population Structure I. BARRAI, '2 A. RosITO,1 G. CAPPELLOZZA,3 G. CRISTOFORIj4 C. VULLO,4 C. SCAPOLI, I AND G. BARBUJANI1 SUMMARY The allele frequencies for beta-thalassemia for 51 localities in the province of Rovigo, and in 25 localities in the province of Ferrara, were studied. It was observed that in the province of Ferrara there is a significant dine of frequencies; these decrease from the coast of the Adriatic Sea toward the west. No such gradient was visible in Rovigo. It was advanced, also on the basis of geography documented by ancient maps, that in the province ofRovigo there were multiple foci of selection for the thalassemia gene, and that in the province of Ferrara selection was stronger in the Oriental part of the area. Examination of the isolation by distance model with these data showed that the Malecot-Morton model fits for the Ferrara data and geography, whereas it does not for Rovigo. INTRODUCTION The frequencies of beta-thalassemia in the Po Delta in Northern Italy have been studied by several investigators in the past 30 years (see [1] for references); in the same area, particularly in the part of the Delta belonging to the province of Ferrara, the frequencies of other genetic markers have been described [2, 3]. The effect of genetic counseling and of medical genetics procedures on the incidence of marriage between beta-thalassemia heterozygotes [4, 5] and on the incidence of Cooley anemia was also investigated [6, 7]. Models of genetic structure of Received December 20, 1983; revised April 4, 1984. This study was done with the assistance of the MPI 60% and 40% funds. 1 Institute of Zoology, University of Ferrara, Ferrara, Italy. 2 Interdisciplinary Center of the National Academy of the Lincei, Rome, Italy. 3Microcitemia Center, USL 30, Rovigo, Italy. 4 Pediatric Division, USL 31, Ferrara, Italy. C) 1984 by the American Society of Human Genetics. All rights reserved. 0002-9297/84/3605-0017$02.00 1121 1122 BARRAI ET AL. the population were tested using markers of the ABO and Rh systems and markers of the GLO, PGP, DIA, ESD, GPT, and 6PGD enzyme systems [8-10]. We have available now the data on the frequencies of microcythemia in the whole delta of the river, in the provinces of Ferrara and Rovigo (fig. 1). We can study now the geographical variation of frequencies inside the delta area and compare the population residing at the north of the river (Rovigo) with the pop- ulation at the south (Ferrara). Since it was observed that a gradient of frequencies exists for Ferrara with distance from the sea [ 1, 9], we want to study here if such a gradient is confirmed by the present set of data and also if it exists for the area north of the river. We shall study further the population structure for the two provinces separately, using the thalassemia gene only; for Ferrara, we shall be able to compare the structure obtained with markers that seem to be neutral at present with the structure of a gene that is still lethal in homozygous state. MATERIALS AND METHODS In the Rovigo province, blood samples were collected from 14,806 children in elementary schools in 51 residential areas or Comunes in the years 1979, 1980, and 1981; the het- erozygous state for beta-thalassemia was diagnosed by determination of hemoglobin A2 level. Out of 14,806 individuals, 1,159, or 7.82%, were found to be beta-thalassemia heterozygotes from the hemoglobin A2 test (table 1). In the Ferrara province, screening for microcythemia was initiated many years before Rovigo; here, we consider the data from 61 screening campaigns in elementary schools of 24 Comunes and 1 sub-Comune (Renazzo). The screenings refer to a period from 1957 to 1975 (tables 2 and 3). The methodology for screening was the Simmel test, which detects increased osmotic resistance and which is an indicator, in this area, of the hetero- ADIGE ROVER R o vo~~~~~~~~~~~~~~.i g 2r. 0 FIG. 1.-The provinces of Ferrara and Rovigo in the Po Delta in Italy. Rovigo is a narrow belt limited by the river Adige at the north, and by the Po at the south. BETA-THALASSEMIA 1123 TABLE 1 LOCALITIES, SAMPLE SIZES, AND No. THALASSEMIC HETEROZYGOTES IN THE PROVINCE OF RovIGO Locality Sample size Heterozygotes DS X Y Adria ....................... 938 61 22 86 80 Ariano Polesine .............. 265 18 22 93 68 Arqua Petrarca ............... 188 14 48 62 75 Badia Polesine ............... 754 49 66 44 85 Bagnolo Po .................. 88 6 66 42 75 Bergantino ................... 190 9 85 24 80 Bosaro ...................... 81 6 46 65 74 Calto. ....................... 58 5 73 32 73 Canaro ...................... 188 18 54 57 67 Canda ....................... 73 4 65 45 76 Castel Guglielmo ............. 143 10 63 46 77 Castelmassa ........... 348 25 80 28 76 Castelnovo Bariano ........... 170 12 83 26 77 Ceneselli .................... 180 21 76 33 75 Ceregnano ................... 237 26 38 72 79 Contarina .................... 371 30 11 100 77 Corbola ..................... 84 10 22 89 74 Costa di Rovigo .............. 199 7 50 58 67 Crespino . ............ 157 6 38 74 72 Donada ...................... 318 37 11 99 78 Ficarolo ..................... 191 19 72 38 68 Fiesso ....................... 295 23 60 51 70 Frassinelle ................... 129 8 51 56 72 Fratta Polesine ............... 218 20 55 55 77 Gaiba ....................... 72 6 69 42 68 Gavello . ............ 92 9 33 76 76 Giacciano con Baruchella ...... 151 12 69 39 81 Guarda Veneta ............... 74 12 43 67 72 Lendinara .................... 941 80 58 51 83 Loreo ....................... 196 19 12 98 81 Lusia. ....................... 270 25 52 56 85 Melara ...................... 135 2 91 19 81 Occhiobello .................. 642 49 62 50 65 Papozze ..................... 55 5 26 86 72 Pettorazza ................... 86 2 27 82 89 Pincara ...................... 118 12 57 52 73 Polesella ..................... 219 24 48 63 70 Pontecchio ................... 127 9 42 67 76 Portotolle .................... 521 64 10 109 69 Rosolina ..................... 294 27 7 102 82 Rovigo ...................... 2,985 213 43 66 82 Salara ....................... 79 8 72 37 73 S. Bellino ................... 81 8 58 50 68 S. Martino Venezze ...... ..... 286 22 35 72 88 Stienta ...................... 198 17 65 47 67 Taglio di Po .................. 429 22 15 99 75 Trecenta ..................... 211 13 70 40 77 Villadose .................... 412 36 35 74 82 Villamarzana ................. 89 6 51 58 75 Villanova del Ghebbio ......... 123 8 55 54 80 Villanova Marchesana ......... 57 5 31 80 75 NOTE: DS is the distance of the locality from the coast; X and Y, the linear coordinates in centimeters on a map of the area. 1124 BARRAI ET AL. TABLE 2 LOCALITIES, SAMPLE SIZES, AND No. MICROCYTHEMICS IN THE PROVINCE OF FERRARA Locality Sample size Microcythemics DS X Y Argenta ................ 3,140 304 33 70 32 Berra ................... 1,193 128 30 80 74 Bondeno ................ 1,874 85 66 36 61 Cento .................. 2,097 51 75 27 44 Codigoro ............... 3,246 387 13 91 55 Comacchio .............. 4,527 317 5 98 39 Copparo ................ 7,072 645 38 69 62 Ferrara ................. 25,709 1,838 50 52 56 Formignana ............. 730 81 33 71 56 Iolanda di Savoia ........ 1,701 159 25 81 61 Lagosanto .............. 1,686 170 9 94 48 Masi Torello ............ 383 47 36 65 51 Massa Fiscaglia ......... 1,718 203 20 83 53 Mesola-Goro ............ 3,109 346 5 105 60 Migliarino-Migliaro ...... 2,239 234 25 79 50 Mirabello ............... 180 8 62 40 55 Ostellato ................ 3,019 306 24 77 46 Poggio Renatico ......... 1,018 53 60 42 48 Porto Maggiore .......... 2,795 233 35 67 40 Renazzo ................ 1,582 43 76 28 48 Ro Ferrarese ............ 397 49 43 63 68 S. Agostino ............. 389 9 68 34 51 Tresigallo ............... 925 63 28 74 54 Vigarano Mainarda ....... 1,600 91 60 43 56 Voghiera ............... 654 45 40 62 48 NOTE: DS is the distance of the locality from the coast; X and Y, the linear coordinates in centimeters on a map of the area. zygous state for beta-thalassemia. Out of72,983 individuals tested in 61 screenings, 5,895 were positive for the test, resulting in a frequency of microcythemia of 8.08%. We have discussed elsewhere the characteristics of the Simmel test as an indicator of the heterozygous state in this area [11]; however, in table 4, we give the data of a run of 1,102 tests on apparently normal individuals tested in 1978 [12]. Assuming that the diagnosis based on the level of hemoglobin A2 is the true indicator of the heterozygous state for beta-thalassemia, the Simmel test gives a high rate of false positives (about 10%) and of false negatives (about 1.7%). Out of 100 microcythemics so detected, 90 are probably heterozygous for beta-thalassemia; the others may be sideropenic individuals, alpha-thalassemia hetero- zygotes, and double heterozygotes for alpha- and beta-thalassemia. Furthermore, in the population at large, there may be as many as three heterozygotes per thousand individuals TABLE 3 No. SCREENING BY COMUNE IN THE FERRARA PROVINCE, 1957- 1975 No. screening Comunes 1 .......... 4 2 .......... 10 3 ......... 7 4 .......... 4 Total .......... 25 BETA-THALASSEMIA 1125 TABLE 4 CORRELATION BETWEEN THE SIMMEL TEST AND THE LEVEL OF HEMOGLOBIN A2 IN 1,102 INDIVIDUALS FROM THE FERRARA POPULATION HEMOGLOBIN A2 SIMMEL TEST + - TOTAL + ...... 182 20 202 - ...... 3 897 900 Total ...... 185 917 1,102 who go undetected as false negatives. The estimates of heterozygote frequencies through the Simmel test may then be inflated by an 8% bias in this population; the overall estimate of 8.08% from these data may be as low as 7.30%. Under the above restrictions, here we shall consider the rates of individuals with raised hemoglobin A2 in Rovigo, and the rates of individuals with increased osmotic resistance in Ferrara, as estimates of rates of heterozygosis for beta-thalassemia.